Mark Wefers Bettink

Measuring mitochondrial oxygenation and respiration in vivo in a human endotoxemia model 6 111 in the control group (Fig. 2A). These time points were chosen to match the start of the experiment (baseline), the peak of cytokine release (1.45 hours after LPS) the peak in temperature rise (4 hours after LPS) and the end of the experiment (7 hours after LPS). All COMET measurements were performed in the supine position. At baseline the volunteers were rested supine for at least 30 min after venous and arterial puncture to minimize the effect of stress on the baseline measurement. Measurement of the mitoPO 2 and mitoVO 2 were performed by the first author on both locations. The measurement probe was held above the ALA treated skin by hand. Occlusion of the microcirculation in the skin was achieved by manual firm pressure with the measurement probe (Fig. 2B). This simple procedure repeatedly created a measurable mitoVO 2 , due to cessation of the microvascular oxygen supply and ongoing cellular oxygen consumption. The mitochondrial oxygen concentration was measured before and during application of pressure at an interval of 1 Hz, using two laser pulses per measurement. The mitoVO 2 is analyzed using Michaelis-Menten kinetics (Fig. 4). We previously described these principles in detail and provided a working implementation of the technique for mitoVO 2 measurements[16]. 2.B 2.A Fig 2. COMET measurements. A. Dynamic measurement with COMET setup in the experiment. Microcirculation of subject’s skin was stopped by direct pressure with the measurement probe by the researcher. B. COMET monitor display showing an ongoing dynamic measurement of mitoVO 2 . Raw data of COMET monitor was captured from the COM-port using a laptop. Photograph shown with permission of subject. Statistics Data are represented as mean and standard deviation or median with range/interquartile range based on their distribution (assessed using the D’Agostino & Pearson omnibus